RESUMEN

BACKGROUND: Respiratory syncytial virus (RSV) bronchiolitis is a seasonal disease that has an enormous burden on health systems across the world. RSV disease manifestations in children range from mild upper respiratory tract infections to severe lower respiratory tract infections, including pneumonia and bronchiolitis. In South Africa, the seasonality of RSV disease causing both upper and lower respiratory tract illness is well documented. OBJECTIVES: To describe the incidence of RSV bronchiolitis among patients ≤24 months of age who presented to a tertiary institution with a diagnosed viral bronchiolitis over a 4-year period. Secondary aims included determining: (i) the risk factors for the development of RSV bronchiolitis; (ii) the fatality rates and risk factors associated with mortality; (iii) the correlation with c-reactive protein values and risk of comorbid bacterial infection; and (iv) the impact of seasonality on RSV incidence. METHODS: A retrospective chart-based analysis of laboratory-confirmed RSV cases in children ≤24 months, presenting to Steve Biko Academic Hospital from January 2013 to December 2016, was undertaken. Epidemiology, risk factors and local weather data were collected as part of the analysis. RESULTS: During the 4-year period, a total of 1 127 nasopharyngeal aspirates (NPAs) was collected. RSV was isolated from 162 NPAs by either immunofluorescence (84%) or polymerase chain reaction (16%). Of the 162 patients with RSV bronchiolitis, 131 (80.9%) had a known HIV status. Only 2 (1.5%) of the patients whose status was known were HIV-infected; 26 (19.8%) were HIV-exposed and confirmed negative; and 103 (78.6%) HIV-unexposed. Forty-nine patients (30.2%) with RSV required intensive care unit (ICU, either paediatric or neonatal) admission. Thirty-four (69.4%) of these were <6 months old. Prematurity (27.8%) and cardiac lesions (13%) were the most common risk factors for acquiring the disease identified in patients with RSV bronchiolitis. CONCLUSION: RSV is still a commonly detected virus among infants who are admitted for bronchiolitis. Significant risk factors associated with admission due to RSV bronchiolitis were prematurity, being <6 months of age and congenital cardiac disease. Male gender and HIV status did not appear to increase the risk of RSV bronchiolitis. In fact, HIV seems to have a protective effect against specifically RSV bronchiolitis in children <2 years of age. Young babies, especially premature infants with RSV bronchiolitis, are at considerable risk of requiring ICU admission, which leads to a significant increase in admission costs.

Asunto(s)

RESUMEN

Differentiation of hematopoietic stem cells into B lymphocytes requires the concerted action of specific transcription factors, such as RUNX1, IKZF1, E2A, EBF1 and PAX5. As key determinants of normal B-cell development, B-lineage transcription factors are frequently deregulated in hematological malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and affected by either chromosomal translocations, gene deletions or point mutations. However, genetic aberrations in this developmental pathway are generally insufficient to induce BCP-ALL, and often complemented by genetic defects in cytokine receptors and tyrosine kinases (IL-7Rα, CRLF2, JAK2 and c-ABL1), transcriptional cofactors (TBL1XR1, CBP and BTG1), as well as the regulatory pathways that mediate cell-cycle control (pRB and INK4A/B). Here we provide a detailed overview of the genetic pathways that interact with these B-lineage specification factors, and describe how mutations affecting these master regulators together with cooperating lesions drive leukemia development.

Asunto(s)

RESUMEN

Drug-induced phospholipidosis is marked by an excessive accumulation of phospholipids in lysosomes which can occur after exposure to cationic amphiphilic drugs. Phospholipidosis is considered as an adverse side effect and may delay or negatively affect registration of drug candidates. Currently, the gold standard method of phospholipidosis detection is electron microscopy on tissue samples. This technique is time consuming and only performed relatively late in drug development. Therefore, in vitro screening methods for phospholipidosis are essential in early drug development. In this study, an in vitro phospholipidosis detection assay is developed with CHO-K1 and HepG2 cells by using the fluorescent marker NBD-PE and high content screening analysis. Lysosomal localization of NBD-PE was demonstrated by colocalization with Lysotracker and lamellar body formation by electron microscopy. Upon drug exposure, lysosomal NBD-PE accumulation can be visualized and quantified. Validation with 56 reference compounds, divided in 25 phospholipidosis inducers and 31 negative compounds, showed that this new in vitro assay has a high sensitivity (CHO-K1=92.0% and HepG2=88.0%) and specificity (CHO-K1=87.1% and HepG2=80.6%) for predicting phospholipidosis in vivo. Thus a selective screening tool has been developed for early selection of drug candidates with low probability for phospholipidosis.

Asunto(s)

RESUMEN

A 28-year-old woman who was treated for vaginal candidiasis with Gyno-Daktarin vaginal capsules (miconazole nitrate) became pregnant because a condom used during intercourse had ruptured. Incubation in vitro with 400 and 1200 mg miconazole nitrate vaginal capsules (Gyno-Daktarin 3 and Gyno-Daktarin I), but not miconazole nitrate vaginal cream (20 mg/g), was shown to damage rubber condoms. Patients using vaginal medicines should be aware of a possible adverse effect on rubber condoms or contraceptive diaphragms and a subsequent increased risk of pregnancy or contagious diseases such as AIDS. Fatty excipients such as glycerin, paraffin, petrolatum or Witepsol may be involved.

PIP: A 28-year-old woman who was treated for vaginal candidiasis with Gyno-Daktarin vaginal capsules (1 capsule per day containing 400 mg miconazole nitrate for 3 days) became pregnant because a condom used during intercourse on the 3rd day had ruptured. The pregnancy was later was terminated by vacuum aspiration. With the collaboration of Janssen Pharmaceutica the TNO Plastics and Rubber Institute studied the effect of Gyno-Daktarin on the quality of condoms. Three different forms were investigated: Gyno-Daktarin cream (20 mg miconazole nitrate/g), Gyno-Daktarin in 1 vaginal capsule (1200 mg miconazole nitrate), and Gyno-Daktarin in 3 vaginal capsules (400 mg miconazole nitrate). Before the investigation six different types of condoms were selected from among the commonly sold types in the Netherlands. The condoms were brought into contact with the above three products. Then they were exposed for one hour to a temperature of 37 degrees Celsius and cooled off to room temperature. Among 450 condoms that were handled in this fashion (75 per brand) length changes, bursting volume, and bursting strength measured by means of ISO 4074, the valid standard of the International Standard Organization, were compared to the outcome of 600 condoms (100 per brand) that had not been exposed to Gyno-Daktarin. The three different combination forms of Gyno-Daktarin were each tested on 150 condoms (25 per brand). There was no change in the length of condoms that were brought into contact with the cream (20 mg/g), however, after incubation in vitro with both 400 and 1200 mg miconazole nitrate vaginal capsules (Gyno-Daktarin 3 and Gyno-Daktarin 1) the length was augmented by an average of 20%. After treatment with the two kinds of capsules the bursting pressure and the bursting volume were reduced by an average 35-44%. Patients using vaginal medicines should be aware of a possible adverse effect on rubber condoms or contraceptive diaphragms and a subsequent increased risk of pregnancy or contagious diseases such as AIDS.

Asunto(s)

RESUMEN

Chylomicron remnant catabolism appears to be mediated by apolipoprotein (apo) E binding to hepatic lipoprotein receptors. Previously, the apo B,E(LDL) receptor and a unique apo E-binding protein (referred to as the apo E receptor) were isolated from solubilized canine and human livers. In the present study, the apo E-binding fraction was further characterized and found to contain at least three proteins, all of which bind apo E-containing lipoproteins with high affinity. The 56-kDa band was found to contain the alpha- and beta-subunits of F1-ATPase, presumably derived from mitochondrial membranes. In addition, an apo E-binding protein with an apparent Mr approximately equal to 59,000 was identified. The 59-kDa protein displays calcium-independent binding on ligand blots, but displays both calcium-dependent and -independent binding in assays performed with detergent-solubilized protein. The 59-kDa protein recognized lipid-free as well as lipid-bound apo E in ligand blots, and also bound apo E-2, apo E-3, and apo E-4 in a comparable way. Monoclonal antibodies produced against the 59-kDa protein did not react with the 56-kDa proteins. Normal human liver, as well as the liver of a patient lacking the apo B,E(LDL) receptor, possessed the 56-kDa and 59-kDa proteins. These data indicate that liver cells possess at least three proteins, in addition to the apo B,E(LDL) receptor, that bind apo E-containing lipoproteins with high affinity. The physiological role of these proteins in apo E metabolism remains to be determined.

Asunto(s)

RESUMEN

Low-density lipoprotein receptors from adult human liver and the human hepatoblastoma cell line HepG2 were analyzed by polyacrylamide electrophoresis in SDS followed by immuno- and ligand blotting. In both liver and HepG2 we detected a protein band with apparent relative molecular mass of 130 kDa, which is similar to that of the LDL receptor in fibroblasts. In addition we showed that HeLa cells also possess this LDL-receptor protein.

Asunto(s)

RESUMEN

We have previously identified a patient with familial hypercholesterolaemia (FH), where the defect appears to be caused by a deletion in the 3' region of the low-density lipoprotein (LDL)-receptor gene. We have now isolated the LDL-receptor gene from the patient and have studied the defect at the DNA level. Restriction mapping and sequence analysis demonstrate that a 4-kb DNA deletion has occurred between two alu-repetitive sequences that are in the same orientation, one in intron 12 and the other in intron 14. This deletion eliminates exons 13 and 14, and changes the reading frame of the resulting spliced mRNA such that a stop codon is created in the following exon. Immuno- and ligand-blot analysis using cultured fibroblasts from this patient revealed the normal gene product, but failed to detect any smaller receptor protein. This implies that the truncated receptor protein that is synthesised is rapidly degraded. We suggest that in this patient the deletion is caused by an unequal crossing-over event that occurred between two homologous chromosomes at meiosis.

Asunto(s)

RESUMEN

A new method for the apolipoprotein E phenotyping has been developed. The method is based on isoelectric focusing of either delipidated or guanidine-HC1-treated serum or plasma in a horizontal slab gel system followed by immunoblotting using either polyclonal or monoclonal anti-apolipoprotein E antibodies as first antibody. Apolipoprotein E phenotyping with this method in 200 serum samples that had been stored at -20 degrees C for more than one year gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated from fresh serum followed by protein staining. Compared with the conventional method, the present method is less laborious because ultracentrifugation to isolate VLDL is not needed; it is suitable for large scale screening purposes; it needs only a few microliters of serum or plasma, and can easily be performed with samples with low concentrations of apolipoprotein E.

Asunto(s)

RESUMEN

The major glycoprotein of pancreatic zymogen granule membranes (GP-2) was detected in the medium of acinar cell suspensions from rat pancreas. Its release from the cells was studied in pulse-chase metabolic labeling experiments with radioactive methionine. GP-2 (apparent Mr = 80 000) was found to be processed to a form of slightly lower apparent Mr (75 000) after about 4 h chase. At about the same time this smaller form of GP-2 appeared in the medium. These results are in accordance with earlier findings in vivo. At different chase times acinar cells were extracted with Triton X-114 to separate water-soluble proteins from membrane-associated (hydrophobic) proteins. This experiment showed that GP-2 is slowly converted from a membrane-bound glycoprotein to a soluble glycoprotein after its reduction in apparent molecular mass, causing its detachment from the membrane. Further analysis indicated that the detachment process may occur at the zymogen granule membrane as well as the plasma membrane. Immunocytochemistry on ultrathin cryosections of pancreatic tissue showed that GP-2 is localized on zymogen granule membranes, plasma membranes and in the acinar lumen. Although in much smaller quantities, GP-2 is also present in the granule content. Thus, in summary, GP-2 is synthesized as a true membrane glycoprotein which is gradually processed to a soluble species and is found in the secretion.

Asunto(s)

RESUMEN

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP-2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse-chase experiments were performed. The incorporation of the first newly synthesized GP-2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP-2 reaches the cell surface within the same time span. After 6-8 h chase considerable more newly synthesized GP-2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP-2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane. GP-2 is the only protein that appears in discernable quantity in the plasma membrane during 1-4 h after a pulse label. Nevertheless GP-2 comprises only a small percentage of externally 125I-iodinated plasma membrane proteins. We conclude that GP-2 has a high turnover rate at the plasma membrane level. Treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP-2.

Asunto(s)

RESUMEN

The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight Mr = 73000. Within 60 min after synthesis it is almost completely converted to the mature form (Mr = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-beta-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides. Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5-6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73000-61000. We conclude that GP-2 contains five or six N-linked carbohydrate chains. From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (greater than 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (less than 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.

Asunto(s)

RESUMEN

High levels of pancreatic ribonucleases are found in ruminants, species that have a ruminant-like digestion and several species with coecal digestion. Pancreatic ribonucleases from several independently evolved species with ruminant-like digestion were investigated to test a hypothesis that glycosylation of ribonucleases may have some function in species with coecal digestion and that glycosylation of the enzyme may not be advantageous for ruminants. Ribonucleases from the hippopotamus, two-toed sloth and three-toed sloth were isolated by extraction with sulfuric acid and affinity chromatography. Complete amino acid sequences were determined for the ribonucleases from the hippopotamus and two-toed sloth and a partial sequence for the enzyme from the three-toed sloth. The amino acids 75-78 of hippopotamus ribonuclease were positioned by homology with other artiodactyl ribonucleases. In hippopotamus ribonuclease a heterogeneity was found at position 37, half of the molecules containing glutamine acid the other half lysine. Hippopotamus ribonuclease differs less from pig and bovine ribonuclease than these differ from each other, because more ancestral characteristics have been retained. Although hippopotamus ribonuclease contains all four Asn-X-Ser/Thr sequences previously found to be glycosylation sites in one or more pancreatic ribonucleases, only the sequence Ans-Met-Thr (34-36) is glycosylated in the variant with glutamine at position 37, while the variant with lysine at this position is carbohydrate-free. Both sloth ribonucleases are completely glycosylated at the sequence Ans-Met-Thr (34-36) with a simple type of carbohydrate chain. The amino acid sequence of two-toed sloth ribonuclease shows some interesting coupled replacements.

Asunto(s)